Summary: Our laboratory has used the SCID mouse lethality model to demonstrate neutralization of vaccinia virus with VIG and VIGIV. We have shown that 5 mg of VIGIV (but not VIG) can neutralize 106 vaccinia organisms (Wyeth strain), when co-incubated with virus, and injected into SCID mice. In a preliminary experiment, using two in vitro high-titer lots of IGIV we showed that the selected IGIV's delayed mouse mortality, similar to VIGIV. In these experiments, VIGIV was given at the 5 mg dose, but was diluted into a large volume (4 times volume used in conventional neutralization experiments), in order to control for the volume of IGIV needed to achieve the same dose-equivalent of anti-vaccinia antibodies as in VIGIV (e.g. 40 mg IGIV/mouse). Neither solution demonstrated complete protection, suggesting that at low concentrations of antibody, neutralization was less effective. IGIV-mediated disease delay was unaffected by dialysis of the starting material, indicating that artifacts due to excipients are unlikely. We have recently compared IGIV's (high and low titer) to VIGIV (in diminishing doses), in SCID mice infected with 105 PFU of virus. The results suggested good efficacy of IGIV's against this low dose viral challenge. In a second experiment, mice have received a higher viral dose, for comparison with VIGIV, results are pending. All results are compared to in vitro assays, for each experiment. If IGIV lots appear to have similar efficacy to VIGIV, studies will be performed to determine whether these preparations are capable of providing prophylaxis (pre- and post-exposure), and treatment against disseminated vaccinia in SCID mice. IGIV distribution and half-life have already been described in SCID mice, which will enable reasonable estimates of timing. To more closely approximate the human situation, the mice will be infected by scarification or intradermal vaccinia injection, after dose-ranging studies for vaccinia to determine the optimal testing dose. If IGIV is effective, this will provide additional data to support the notion that IGIV could be a useful backup in the event of depletion of VIG/VIGIV supplies. An opposite finding could accelerate efforts to increase the supply of specific VIGIV. The function and efficacy of anti-vaccinia antibodies may be different in IGIV compared to VIG products, because the donors were vaccinated in the distant past. Any differences in antibody subclass or affinity between anti-vaccinia antibodies in IGIV and VIG products would have potential clinical consequences. Such findings would also inform efforts to produce more potent VIG product. We plan to study antibody subclass and affinity in high-titer IGIV and VIGIV. Antibody subclasses have been enriched (using protein A sepharose columns) from selected high-titer IGIV lots, as well as VIGIV. Our laboratory is experienced in subclass isolation. Each subclass will be tested for neutralizing ability by PRN assays, B-gal assays, and in vivo neutralization in SCID mice. Antibody affinity will be assessed using the Biacore. A Biacore chip will be coated with identified neutralizing epitopes for vaccinia virus, by conventional amine coupling. The chip will be tested against VIG products, and IGIV. Recent work by others has shown the feasibility of detecting epitope binding using polyclonal human serum. Positive controls, in addition to VIG products, will include monoclonal antibody preparations if available, against vaccinia epitopes. Negative controls will include low-titer IGIV, serum from unvaccinated individuals, and vaccinia-adsorbed IGIV. Initial experiments will focus upon characterizing conditions (e.g. concentration of antigen on chip, flow rates, optimal serum/IGIV concentrations for detection of specific binding). Interaction curves (refractive units) will be generated and compared. It is hoped that the reaction will follow "pseudo first order" kinetics, enabling calculation of accurate affinity constants.